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Clinical Microbiology of the Dengue Virus

INTRODUCTION The dengue virus causes dengue and dengue hemorrhagic fever. It is an arboivirus, within thia group it is from the family Flavivirade, which includes hepatitis c, West Nile, yellow fever and Japanese and St. Louis encephalitis. All these viruses are spread by mosquitoes. The two types of mosquitoes that spread dengue are Aedes aegypti and Aedes albopictus, Aedes aegypti being the most common. The virus itself is composed of single-stranded RNA and has four serotypes. These sertotypes are DEN-1, DEN-2, DEN-3 and DEN-4.

The transmission cycle of dengue begins with a dengue infected person. The person will have the virus circulating in their blood, a virenia that lasts for about five days. During this period, an uninfected female Aedes Aegypti mosquito bites the person and ingests the blood. Within the mosquito, the virus replicatesduring an extrimsic incubation period for eight to 12 days. The mosquito then bites a person, which gives the virus to that person and any other person it bites during its lifetime.

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The virus then replicates in that person and produces symptoms, which begin to appear on an average of four to seven days, but can range from three to 14. This is known as an intrinsic incubation period. The usual signs of dengue are a fever of 103 to 105 degrees, severe headache, pain behind the eyes, body aches and pains, rash on the skin, and nausea or vomiting. Sometimes bleeding occurs. The usuaul spots for bleeing are the nose, gums or skin. Rarely, the patient suffering of dengue will develop shock, which is known as dengue shock syndrome.

More that 40% of the world’s population is at risk for infection from it and 1. 5 million people are treated each year for dengue fever and dengue hemorrhagic fever. The first reports of major epidemics of illnesses thought to be dengue only occurred on three continents. The three continents were Asia, Africa and North America and they occurred in 1779 and 1780. However, some believe that there were epidemics dating back to the Chin Dynasty of China, which was in power from 265 to 420 A. D.

Dengue has reemerged over the last 20 years and has come back with an expanded geographic distribution of both the viruses and the mosquito vectors and has increased epidemic activity. Demographic, societal and public health infrastructure changes during this period have contributed to this. VECTOR CONTROL Dengue is tramsitted by an infected female mosquito. Aedes aegypti is primarily a daytime feeder and mainly bites in the morning or in the late afternoon in covered areas. It is not usuallly found in tropical forests, except in Africa.

The female would rather lay her eggs in artificial containers than natural ones in fairly clean water and areas that are close to human habitation. The Aedes aegypti had been mostly eradicated during the 1950s, ’60s and most of the ’70s due to the spraying of DDT and other pesticides. The eradication program was discontinued during the ’70s, so this species of mosquito began to reinvade the areas and countries from which it had been eradicated. By the ’90s, Aedes aegypti had essentially regained all of, the land it held before the eradication program began.

During the ’80s, the Americas began experiencing major epidemics of the disease in countries that had not any for 35 to 130 years. Therefore, there is now a strong emphasis on having a proactive surveillance system. The objective of which is to provide early and precise information on the time, location, virus serotype and disease severity. The analysis of these four aspects are the key that is needed to predict dengue transmission and to guide implementation of control measures long before the transmission of dengue is at its peak.

For exmaple, there is a dengue surveillance system in place in Puerto Rico, which provides a system in which blood samples are taken by physicians, health centers, private and public hospitals and privates laboratories in Puerto Rico, are transported to the Dengue Branch and then tested by the CDC. The analysis is provided to various groups and individuals through press releases, articles in newspapers and scientific journals in an attempt to educate the public so that they know where the outbreaks are and how to help control them. The use of larvicides and incesticides are still widely used as well, but are not always very effective.

Using larvicide requires placing chemicals into containers to kill the mosquito lavae. Ultra-low volume (ULV) spraying of incesticides is also used quite frequently to kill adult mosquitoes. This method uses machines that make very small particles of incesticide, which are then sprayed and then the wind currents carry the particles to the desired locations. ULV machines are usually mounted on trucks or are portable machines that are carried to the fields by workers and then used. However, the incesticide has to come in direct contact with the mosquito in order to kill it.

Since the Aedes Aegypti usually lives in houses, and usually secluded locations in the house such as closets, the incesticide very rarely comes in the direct contact with the mosquito that it needs to to take effect. Therefore, this method is usually ineffective and usually expensive as well. Once the mosquitoes are in ones house, one can use commercial aerosol sprays to kill those that are found. However, a common recurring theme is that the mosquitoes are building a tolerance to the sprays and they are devloping “knockdown resistance. ” This means that when you spray the mosquitoes, it paralyzes them, knocking them to the floor.

The effect is temporary and when it wears off, they fly away unharmed. Therefore, it you use these sprays, you want to squash the mosquitoes once they have been knocked down. Other vector controls being used are biological and environmental controls. Biological controls are largely experimental and involve using small fish or copepods, small invertebrate crustaceans, that feed on first and second-stage mosquito larvae. Environmental controls being used eliminate or control the habitats of the larvae where the mosquito eggs are layed and the immature mosquitoes develop.

Including, but not limited to, emptying water from containers or covering containers that are being used, disposal of containers that are not being used and improving water supplies so that the storing of water will not be as necessary. Because chemical control can only be used in certain situations and because bioloigical control is still largely experimental, environmental control is the going to be the most effective for long-term control of Aedes aegypti. DIAGNOSIS There are several indicators that somebody has dengue fever.

One would look for increased blood pressure, both level and pulse pressure, evidence of bleeding in the skin or other sites, hydration status, evidence of increased vacular permeability as evidence by plueral effusions or acites and perform a tourniquet test. A tourniquet test consists of inflating the blood pressure cuff to a point midway between systolic and diastolic pressure for five minutes. After deflating the cuff, wait for the skin to return to its normal color and then count the number of petechiae visible in a one-inch square area on the ventral surface of the forearm.

The results of a positive test will show 20 or more petechiae per 1 inch squared or 6. 25 cm squared. (see attachment A) Laboratory tests that are used include CBC. In patients with dengue, the leukocyte counts are often low and the patient may even be neutropenic. Platelet levels should also be checked and pne should do serial hematocrits for hemoconcentration. Other useful tests are serum albumin and protein, liver function tests and urine analysis to check for microscopic hematuria.

Also, there are dengue specific blood tests that can be performed to viral isolation and serology tht are also time specific. A definitive diagnosis of dengue infection can be made only in the laboratory and depends on isolating the virus, detecting viral antigen or RNA in serum or tissues, or detecting specific antibodies in the patient’s serum. An acute-phase blood sample should always be taken as soon as possible after the onset of suspected dengue illness, and a convalescent-phase sample should ideally be taken 2 to 3 weeks later.

Because it is frequently difficult to obtain convalescent-phase samples, however, a second blood sample should always be taken from hospitalized patients on the day of discharge from hospital. Five basic serologic tests have been routinely used for diagnosis of dengue infection; hemagglutination-inhibition (HI), complement fixation (CF), neutralization test (NT), immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (MAC-ELISA), and indirect immunoglobulin G ELISA.

Regardless of the test used, unequivocal serologic diagnosis depends upon a significant (fourfold or greater) rise in the titer of specific antibodies between acute- and convalescent-phase serum samples. The antigen battery for most of these serologic tests should include all four dengue virus serotypes, another flavivirus (such as yellow fever virus, Japanese encephalitis virus, or St. Louis encephalitis virus), a nonflavivirus (such as Chikungunya virus or eastern equine encephalitis virus), and ideally, an uninfected tissue control antigen.

Of the above tests, HI has been the most frequently used; it is sensitive, is easy to perform, requires only minimal equipment, and is very reliable if properly done. Because HI antibodies persist for long periods (up to 48 years and probably longer), the test is ideal for seroepidemiologic studies. HI antibody usually begins to appear at detectable levels (titer of 10) by day 5 or 6 of illness, and antibody titers in convalescent-phase serum specimens are generally at or below 640 in primary infections, although there are exceptions.

By contrast, there is an immediate anamnestic response in secondary and tertiary dengue infections, and reciprocal antibody titers increase rapidly during the first few days of illness, often reaching 5,120 to 10,240 or more. Thus, a titer of = ” src=”/math/12pt/normal/ge. gif” align=baseline1,280 in an acute-phase or early convalescent-phase serum sample is considered presumptive evidence of a current dengue infection. Such high levels of HI antibody persist for 2 to 3 months in some patients, but antibody titers generally begin to wane by 30 to 40 days and fall below 1,280 in most patients.

The major disadvantage of the HI test is its lack of specificity, which generally makes it unreliable for identifying the infecting virus serotype. However, some patients with primary infections show a relatively monotypic HI response that generally correlates with the virus isolated. The CF test is not widely used for routine dengue diagnostic serologic testing. It is more difficult to perform, requires highly trained personnel, and therefore is not used in most dengue laboratories. It is based on the principle that complement is consumed during antigen-antibody reactions.

CF antibodies generally appear later than HI antibodies, are more specific in primary infections, and usually persist for short periods, although low levels of antibodies persist in some persons. It is a valuable test to have in a diagnostic laboratory because of the late appearance of CF antibodies; some patients thus show a diagnostic rise in antibody titers by CF but have only stable antibody titers by HI or ELISA. The greater specificity of the CF test in primary infections is demonstrated by the monotypic CF responses when HI responses are broadly heterotypic; it is not specific in secondary infections.

The CF test is useful for patients with current infections but is of limited value for seroepidemiologic studies, where detection of persistent antibodies is important. The NT is the most specific and sensitive serologic test for dengue viruses. The most common protocol used in dengue laboratories is the serum dilution plaque reduction NT. In general, neutralizing-antibody titers rise at about the same time or slightly more slowly than HI and ELISA antibody titers but more quickly than CF antibody titers and persist for at least 48 years.

Because the NT is more sensitive, neutralizing antibodies are present in the absence of detectable HI antibodies in some persons with past dengue infection. Because relatively monotypic neutralizing-antibody responses are observed in properly timed convalescent-phase serum, the NT can be used to identify the infecting virus in primary dengue infections. As noted above, the HI and CF tests may also give monotypic responses to dengue infection that generally agree with NT results. In cases when the responses are monotypic, the interpretation of all these tests is generally reliable.

In secondary and tertiary infections, determining the infecting virus serotype by NT or any other serologic test is not reliable. Because of the long persistence of neutralizing antibodies, the test may also be used for seroepidemiologic studies. The major disadvantages are the expense, time required to perform the test, and technical difficulty. It is therefore not used routinely by most laboratories. MAC-ELISA has become the most widely used serologic test for dengue diagnosis in the past few years. It is a simple, rapid test that requires very little sophisticated equipment.

Anti-dengue IgM antibody develops a little faster than IgG antibody. By day 5 of illness, most patients (80%) in Puerto Rico whose cases were subsequently confirmed by HI on paired serum samples or by virus isolation had detectable IgM antibody in the acute-phase serum in this assay. Nearly all patients (93%) developed detectable IgM antibody 6 to 10 days after onset, and 99% of patients tested between 10 and 20 days had detectable IgM antibody. The rapidity with which IgM develops varies considerably among patients.

Although the dates of onset are not always recorded accurately, some patients have detectable IgM on days 2 to 4 after the onset of illness whereas others may not develop IgM for 7 to 8 days after onset. This variation is also reflected in the amount of IgM produced and the length of time detectable IgM persists after infection. IgM antibody is produced by patients with both primary and secondary dengue infections and probably by persons with tertiary infections, although the response in some secondary and probably most tertiary infections is low level and transient.

IgM antibody titers in primary infections are significantly higher than in secondary infections, although it is not uncommon to obtain IgM titers of 320 in the latter cases. In some primary infections, detectable IgM persists for more than 90 days, but in most patients, it has waned to an undetectable level by 60 days. A small percentage of patients with secondary infections have no detectable IgM antibody. MAC-ELISA with a single acute-phase serum sample is slightly less sensitive than the HI test with paired serum samples for diagnosing dengue infection.

However, it has the advantage of frequently requiring only a single, properly timed blood sample. In one series of 288 patients during the 1986 epidemic in Puerto Rico, paired blood samples were tested by HI and the single acute-phase sample from the same pairs were tested by MAC-ELISA. The HI test on the pairs indicated that 228 (79%) were considered positive, while MAC-ELISA on the single samples indicated that 203 (70%) were positive. Five samples (1. 7%) showed a false-positive response and 30 samples (10%) showed a false-negative response by MAC-ELISA.

When one considers the difficulty in obtaining second blood samples and the long delay in obtaining conclusive results from the HI test, this low error rate would be acceptable in most surveillance systems. It must be emphasized, however, that because of the persistence of IgM antibody for 1 to 3 months, MAC-ELISA-positive results obtained with single serum samples are only provisional and do not necessarily mean that the dengue infection was current. These results do mean that it is reasonably certain that the person had a dengue infection sometime in the previous 2 to 3 months.

Similarly, a negative result with an acute-phase sample may be a false-negative result because the sample was taken before detectable IgM appeared. Unfortunately, many dengue diagnostic laboratories have adopted MAC-ELISA as a confirmatory test and do not conduct follow-up tests to confirm the presumptive IgM results. As noted above, this may be acceptable for surveillance reports, but it is unacceptable in a clinical setting. If this test is used to make patient management decisions, it could result in a higher case fatality rate among patients with false-negative results.

The specificity of MAC-ELISA is similar to that of HI. In both primary and secondary dengue infections, some monotypic responses may be observed, but in general, the response is broadly reactive among both dengue virus and other flavivirus antigens. With serum samples from patients with other flavivirus infections such as Japanese encephalitis, St. Louis encephalitis, and yellow fever, however, the response is generally more specific; while there may be some cross-reaction with dengue antigens, most specimens show relatively monotypic IgM responses to the infecting flavivirus.

In dengue infections, monotypic IgM responses frequently do not correlate with the virus serotype isolated from a patient. Therefore, MAC-ELISA cannot be reliably used to identify the infecting virus serotype. MAC-ELISA has become an invaluable tool for surveillance of dengue, DHF, and DSS. In areas where dengue is not endemic, it can be used in clinical surveillance for viral illness or for random, population-based serosurveys, with the certainty that any positive results detected indicate recent infections (within the last 2 to 3 months).

A properly timed serosurvey by MAC-ELISA during an epidemic can determine very quickly how widespread transmission has become. In areas where dengue is endemic, MAC-ELISA can be used as an inexpensive way to screen large numbers of serum specimens with relatively little effort. It is especially useful for hospitalized patients, who are generally admitted late in the illness after detectable IgM is present in the blood, but it must be emphasized again that this test should not be used to make patient management decisions.

An indirect IgG-ELISA has been developed that is comparable to the HI test and can also be used to differentiate primary and secondary dengue infections. The test is simple and easy to perform and is thus useful for high-volume testing. The IgG-ELISA is very nonspecific and exhibits the same broad cross-reactivity among flaviviruses as the HI test does; therefore, it cannot be used to identify the infecting dengue virus serotype. However, it has a slightly higher sensitivity than the HI test.

As more data are accumulated on the IgG-ELISA, it is expected to replace the HI test as the most commonly used IgG test in dengue laboratories. A number of commercial test kits for anti-dengue IgM and IgG antibodies have become available in the past few years. Unfortunately, the accuracy of most of these tests is unknown because proper validation studies have not been done. Some evaluations have been published, but the sample sizes have been too small to accurately measure sensitivity and specificity.

Moreover, the samples generally used have represented only strong positives and negatives, with few samples representing optical densities or positive-negative values in the equivocal range. One exception to this were kits that were independently evaluated at CDC; both IgM and IgG test kits had a high rate of false-positive results compared to standard tests, especially with samples with optical densities in the equivocal range. Other studies, however, have given results comparable to those of standard tests.

It is anticipated that these test kits can be reformulated to make them more accurate, making global laboratory-based surveillance for dengue and DHF an attainable goal in the near future. TREATMENT The first candidate dengue vaccines were developed shortly after the viruses were first isolated by Japanese and American scientists. Despite considerable work over the years, an effective safe vaccine was never developed . The World Health Organization designated the development of a tetravalent dengue vaccine a priority for the most cost-effective approach to dengue prevention.

Effective vaccination to prevent DHF will most probably require a tetravalent vaccine, because epidemiologic studies have shown that preexisting heterotypic dengue antibody is a risk factor for DHF. With the support of the World Health Organization, considerable progress in developing a vaccine for dengue and DHF has been made in recent years. Promising candidate attenuated vaccine viruses have been developed and have been evaluated in phase I and II trials in Thailand as monovalent, bivalent, trivalent, and tetravalent formulations.

A commercialization contract has been signed, and the tetravalent vaccine formulation is currently undergoing repeat phase I trials in the United States. Current progress on the live attenuated dengue vaccine has been recently reviewed. Promising progress in the development of alternative vaccine strategies using new molecular technology has also been made in recent years. Recent approaches include the use of inactivated whole-virion vaccines, synthetic peptides, subunit vaccines, vector expression, recombinant live vector systems, infectious cDNA clone-derived vaccines and naked DNA.

The last two approaches appear to be the most promising. An infectious clone of the DEN-2, PDK-53 vaccine candidate virus from Thailand has been constructed, and work is in progress to construct chimeric viruses by inserting the capsid, premembrane, and envelope genes of DEN-1, DEN-3 and DEN-4, into the DEN-2 PDK-53 backbone. Through genetic manipulation, these recombinants may be made to grow better and to be more immunogenic and safer than the original live attenuated virus vaccine candidates. In addition, chimeras are being constructed by inserting he structural proteins of dengue viruses into the infections clones of the 17D yellow fever and the SA14-14-2 Japanese encephalitis vaccine viruses. The development of naked DNA vaccines is in its infancy but shows great promise. This area has been recently reviewed. Despite the promising progress, it is unlikely that an effective, safe, and economical dengue vaccine will be available in the near future. A major problem has been and continues to be lack of financial support for dengue vaccine research.

Thus, other approaches to disease prevention must be developed by using the program components outlined above. BIBLIOGRAPHY http:/www. cdc. gov/ncidod/dvbid/dengue/slideset http:/cmr. asm. org. cgi/content/full/11/3/480? view=long&pmid=9665979 http:www. pubmedcentral. nih. gov/srticlerender. fcgi? artid=379334 http:/www. utmb. edu/discoveringdenguedrugs-together/ http:/searo. who. int/en/section10/section322/section1026. html http://vir. sgmjournals. org/cgi/content/full/87/7/1947


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